Resolved
Authors: Jeremy S. Duffield
Journal: Journal of the American Society of Nephrology (2010)

More than 20 years ago, scientists who were studying fibrosis purified hepatic stellate cells (HSCs) from the liver, separating them by virtue of their distinct cell buoyancy.1 These unusual cells store retinoic acid in health and line the specialized fenestrated capillaries of the liver called hepatic sinusoids and have features in common with pericytes, mural cells of capillaries that have been shown in other tissue beds to be important in sprouting angiogenesis and vascular stability by virtue of cell–cell signaling with underlying endothelial cells.2 When cultured in vitro and activated with serum factors, HSCs generate pathologic collagen matrix and activate intermediate genes involved in migration and contraction, including α-smooth muscle actin (αSMA). It became widely accepted that HSCs were progenitors of the liver myofibroblasts.3 In the kidney, similar pericyte-like cells have been much harder to detect or define. Furthermore, other adherent cell types, including epithelial, endothelial, and myeloid cells, generate collagen matrix and activate expression of the intermediate filament αSMA when cultured in vitro, suggesting that pericyte-like HSC myofibroblast precursors in liver may not exist in other organs and that other cell types have a capacity to become myofibroblasts. Understanding the origin of myofibroblasts (activated spindle or stellate interstitial cells that share features with smooth muscle including expression of αSMA, secreting a pericellular matrix containing collagen and glycosaminoglycans) became more complicated because malignant mammary epithelial cells were shown to migrate through basement membranes and acquire expression of similar intermediate filament proteins to myofibroblasts in vivo.4 The behavior of malignant epithelial cells suggested that epithelium might be a major source of myofibroblasts in nonmalignant diseases. There are now many examples of cultured, nonmalignant epithelial cells acquiring patterns of gene expression in vitro similar to myofibroblasts, providing rationale for the hypothesis that epithelial cells become myofibroblasts in kidney disease.5 Potential markers of this process, called epithelial-mesenchymal transition (EMT), are αSMA and S100A4 (also known as MTS-1 or FSP1), which are expressed in vitro by epithelial cells in response to exogenous TGF-β. Some have used these markers as surrogates for cell lineage analyses in vivo. Although endogenous lineage markers such as CD45 have proved robust and specific for leukocytes, neither αSMA nor S100A4 is a robust marker of lineage, because they lack cell specificity and constancy of expression.6 Indeed, S100A4 in certain tissue beds including injured kidney is detected in macrophages, not myofibroblasts.7–9 Other possible EMT markers include Snail, Twist, Slug, and Id transcription factors, but a range of cell types after injury, including epithelial cells, myofibroblasts, and macrophages, express them.8,10 It is likely that although these transcription factors are important in migration, activation, and differentiation, they do not denote lineage in vivo. Most of the kidney epithelium derives from mesoderm-derived intermediate mesenchyme.11 This process of derivation is termed mesenchymal-epithelial transition (MET), and a reversal of this process has provided a rationale for the hypothesis that epithelium becomes myofibroblasts in kidney injury. In contrast, the epithelium of liver, pancreas, and several other organs derives from endoderm. The absence of developmental MET of other organs, including liver, gives little rationale for EMT events during injury of these organs. Although the in vitro studies are striking, proof that epithelial cells become myofibroblasts in vivo has been limited.12 Indeed, a number of morphologic and experimental tracing studies have failed to demonstrate that epithelial migration and differentiation into myofibroblasts occurs in vivo.8,13,14 With the advent of genomic recombination technology, it has become possible to trace the fate of cells in vivo in mice with confidence. The state-of-the-art method to study EMT in vivo is through lineage tracing, or genetic fate mapping, of cells. This provides an advantage that no cell surface or cytosolic marker has—that is, once a heritable marker is activated in vivo, it is permanent. It will always be detectable in any cell that derives from the labeled cell regardless of differentiation, proliferation, migration, or injury state. Fate mapping of cells in vivo, however, is fraught with pitfalls that are not readily apparent: Genomic recombination leading to stable expression of the heritable marker/reporter (LacZ, Alk-Phos, RFP, GFP, or YFP) must occur only in the cell type to be labeled. For example, the gene locus for Tie2, encoding the endothelial receptor, has been used in studies that label endothelial cells, but Tie2 is expressed transiently by all myeloid precursors, and, thus, the locus effectively drives Cre-mediated recombination in monocytes as well as endothelium.15,16 The gene locus driving expression of the recombinase that denotes cell type specificity should preferably not be activated during disease, because the gene locus used to activate the heritable marker could become active in additional cell types. Recombination must occur efficiently to instill confidence that infrequent events can be detected. the reporter must be robust and have the capacity to be actively expressed at sufficient level to be readily detected in all cell types in the body during health and disease. Recombination of genomic DNA during development is preferable because it occurs at very high frequencies, and developmentally active transcription factors frequently exhibit highly restricted expression. The first studies of epithelial cell lineage after injury were performed in kidney. Iwano et al.12 reported evidence that myofibroblasts in kidney injury derived from proximal tubule cells (PTECs) of the nephron in vivo. They used Cre recombinase driven by a transgene containing part of the γ-glutaryl-transferase promoter, which activates in kidney PTECs neonatally but not developmentally. Mice harboring this transgene were crossed with Rosa26R mice that activate universal bacterial LacZ expression under regulation of the Rosa26 locus promoter after Cre recombination. The authors detected myofibroblasts using antibodies to S100A4 and HSP47 and detected LacZ using antibodies against the gene product β-gal. These studies concluded that >30% of kidney myofibroblasts derive from injured PTECs. These conclusions have been cast into doubt by three recently published comprehensive lineage tracing studies that used three different models of kidney disease and found no evidence of epithelial origin of myofibroblasts.9,10,17 One of these recent studies, however, by using similarly rigorous fate mapping of mesenchyme-derived stromal cells of the developing kidney that mature into perivascular pericytes in the adult kidney, demonstrated that a sizable number of these discrete cells persist in the adult kidney, express PDGFRβ and CD73, and are the primary and probably only source of myofibroblasts that appear during disease of the kidney.10 The new report by Humphreys et al.10 used the loci of developmentally active transcription factors that play key roles in determining mesenchymal cell fate to the epithelial lineage (Six2) or the stromal lineage (Foxd1) to activate two different heritable markers, LacZ or RFP. The gene-targeted mice were either knock-in or BAC transgenic, meaning that the expression of the Cre recombinase faithfully reproduces endogenous gene expression. High levels (>95%) of recombination were achieved in all studies, and the reporters used gave high signal-to-noise outputs when analyzed in tissue sections. Furthermore, all positive and negative controls were presented. The authors studied two well-established models of fibrosing kidney injury. Significantly, the authors used an assay of β-gal enzyme activity to detect cells expressing the LacZ fate marker and native red fluorescence in detection of the RFP fate marker and detected myofibroblasts using directly conjugated anti-αSMA antibodies or Collagen1a1 expression rather than detection of S100A4 (FSP1), which does not identify myofibroblasts in the mouse kidney.8–10 In this study, all kidney epithelia and all stromal cells were labeled and fate mapped. This comprehensive report has been followed by lineage tracing of all epithelial cells of the nephron in the unilateral ureteral obstruction model using the epithelial-specific transgene Ksp-Cadherin-Cre to activate the heritable marker YFP at the Rosa26 locus (R26YFPR).9 Although Ksp-Cadherin is expressed in adult kidney epithelium as well as developing epithelium, its expression is restricted to the epithelial segments of the nephron. Recombination was >90% in all nephron segments except PTECs, where it was 20 to 30%. These authors failed to detect any epithelial cells migrating outside the epithelial compartment, and no epithelial cells became myofibroblasts. In the third study, Koesters and Kriz17 generated a novel kidney disease model in which activation of continuous, Rosa26 locus–driven TGF-β1 expression occurs under the regulation of the epithelial-specific Pax8 promoter using a Tet-inducible system that requires a systemic injection of doxycycline in adult mice for activation. This model leads to progressive tubular atrophy with interstitial fibrosis. All epithelial cells activated to produce TGF-β1 also activated LacZ permanently driven by the Rosa26R locus. Although the labeling of epithelial cells was incomplete and expression of the fate marker LacZ was therefore mosaic, the authors did not identify any interstitial cells expressing the fate marker detected by X-gal blue stain. They concluded that in this TGF-β1–driven disease model, no interstitial myofibroblasts are derived from tubule epithelial cells. Collectively these studies have fate-traced epithelial cells in three different mouse models of kidney disease and failed to detect myofibroblasts originating from epithelium. By contrast, using similar systems, the pericyte origin of myofibroblasts was readily demonstrated.10 These studies should divert investigation of EMT toward the study of pericyte function and biology in tackling fibrotic disease of the kidney. Although there is a lack of developmental rationale for epithelial cells undergoing an EMT process because there is no developmental MET in liver, hepatocyte fate in vivo was reported by Zeisberg et al.18 to include differentiation into myofibroblasts. They used a transgenic model to drive the DNA recombinase Cre under the control of a 2.3-kb fragment of the albumin promoter/enhancer locus to activate the heritable marker LacZ at the Rosa26 locus. Recombination was specific for hepatocytes but was mosaic (approximately 50%). To detect scar-forming myofibroblasts, the authors relied exclusively on expression of S100A4 (FSP1), and to detect the fate marker LacZ, they used antibodies against the gene product β-gal. In a short model of CCl4-liver toxicity and fibrosis, they concluded that as many as 10% of myofibroblasts derive from hepatocytes. Taura et al.19 recently were unable to confirm those findings, using a similar Alb-Cre transgenic model. They also used the Rosa26R, LacZ expressing reporter mouse, but in addition used the Collagen1a1-GFP reporter to identify simultaneously collagen-producing cells in the injured liver and cells derived from hepatocytes. After detecting cell fate by blue stain on the basis of β-gal enzyme activity, they concluded that no myofibroblasts in the CCl4 models of liver disease derive from hepatocytes. Data reported from our laboratory using a 12-week model of CCl4-liver fibrosis are consistent with those of Taura et al.20 Why do similar studies result in such profoundly different interpretations? To the reader, these conflicting reports may be difficult to disentangle. In my opinion, three potential problems exist with the studies of Iwano et al.12 in the kidney and Zeisberg et al.18 in the liver that may have inadvertently led to artifacts. First, the genetic system for the kidney studies used a transgenic Cre driver that may lack cell specificity during disease. Second, the investigators relied on antibody detection of the fate marker bacterial β-gal in tissue sections. Carefully performed experiments indicate that the Rosa26 locus drives adequate expression of LacZ in all cells of adult liver and kidney to detect reliably X-gal blue stain triggered by enzyme activity; this should be the gold standard. In our experience, anti–β-gal antibodies are unreliable and prone to artifacts and need to be compared objectively alongside controls. Third, the marker S100A4/FSP1 has been inappropriately used in the identification of myofibroblasts. Independent studies indicated that S100A4 is expressed in other cells, such as macrophages. In mouse kidney, S100A4 is not detected in any collagen-producing cells.7–9 αSMA, Pdgfrβ, and Coll1a1 all are more reliable markers of the myofibroblast and Pdgfrβ, Coll1a1, and CD73, its precursors. Current comprehensive analyses of epithelial lineage in three models of kidney injury and in CCl4 liver fibrosis indicate that EMT is unlikely to occur in vivo. Rather, they suggest a new paradigm that vascular pericytes are the source of fibrosis-generating myofibroblasts and that epithelial contribution to fibrosis is by paracrine cell signaling rather than cellular transition. These new studies re-focus fibrosis research on microvascular injury rather than epithelial injury. Disclosures None. The Duffield laboratory is funded by National Institutes of Health grants DK73299, DK84077, and DK87389; Genzyme GRIP; American Society of Nephrology Gottschalk awards; and Regulus Pharmaceuticals. I thank Dr. Ben Humphreys for useful discussions.